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UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
UDP-alpha-D-glucose + H-Ras
UDP + D-glucosyl-H-Ras
UDP-alpha-D-glucose + human Ras-GTPase
UDP + D-glucosyl-[human Ras-GTPase]
activity of toxin TcsL in human epithelial colorectal adenocarcinoma Caco-2 cells
-
-
?
UDP-alpha-D-glucose + Rac GTPase
UDP + D-glucosyl-[Rac GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Rac1
UDP + D-glucosyl-Rac1
Rac is the exclusive substrate of the Rho subfamily of GTPases. Rac is a better substrate in the GTDP-bound form than in the GTP-bound form
-
-
?
UDP-alpha-D-glucose + Rac1 GTPase
UDP + D-glucosyl-[Rac1 GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Ral GTPase
UDP + D-glucosyl-[Ral GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Rap GTPase
UDP + D-glucosyl-[Rap GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Ras GTPase
UDP + D-glucosyl-[Ras GTPase]
murine host substrate
-
-
?
UDP-N-acetyl-alpha-D-glucosamine + H-Ras
UDP + N-acetyl-alpha-D-glucosamine-H-Ras
no substrate for wild-type, but substrate for mutant I383S/Q385A
-
-
?
UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
-
-
-
?
UDP-alpha-D-glucose + Cdc42
UDP + D-glucosyl-Cdc42
-
-
-
?
UDP-alpha-D-glucose + H-Ras
UDP + D-glucosyl-H-Ras
good substrate
-
-
?
UDP-alpha-D-glucose + K-Ras
UDP + D-glucosyl-K-Ras
good substrate
-
-
?
UDP-alpha-D-glucose + N-Ras
UDP + D-glucosyl-N-Ras
good substrate
-
-
?
UDP-alpha-D-glucose + Rac1
UDP + D-glucosyl-Rac1
very good substrate
-
-
?
UDP-alpha-D-glucose + RalC
UDP + D-glucosyl-RalC
good substrate
-
-
?
UDP-alpha-D-glucose + Rap2A
UDP + D-glucosyl-Rap2A
good substrate
-
-
?
UDP-alpha-D-glucose + RhoA
UDP + D-glucosyl-RhoA
-
-
-
?
UDP-alpha-D-glucose + RhoB
UDP + D-glucosyl-Rhob
-
-
-
?
UDP-alpha-D-glucose + RhoC
UDP + D-glucosyl-RhoC
-
-
-
?
UDP-alpha-D-glucose + RhoG
UDP + D-glucosyl-RhoG
-
-
-
?
UDP-alpha-D-glucose + TC10
UDP + D-glucosyl-TC10
good substrate
-
-
?
UDP-alpha-D-glucose + TCL signaling G protein
UDP + D-glucosyl-TCL signaling G protein
good substrate
-
-
?
additional information
?
-
UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
-
-
-
?
UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
-
-
-
-
?
UDP-alpha-D-glucose + H-Ras
UDP + D-glucosyl-H-Ras
-
-
-
?
UDP-alpha-D-glucose + H-Ras
UDP + D-glucosyl-H-Ras
Ras is the exclusive substrate of the Ras subfamily of GTPases. Ras is a better substrate in the GTDP-bound form than in the GTP-bound form
-
-
?
additional information
?
-
a circular electron transfer reaction is suggested tha does not directly involve any residues from the toxin. The transfer starts with the split of the glycosidic bond leading to the most stable transient state. The split increases the pK of the phosphoryl oxygen atom, facilitating deprotonation of the accepor, and provides space for the nucleophilic attack
-
-
?
additional information
?
-
lethal toxin from Clostridium sordellii is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases. Lethal toxin selectively modifies Rac and Ras. In Rac, acceptor amino acid is residue threonine 35. No substrates: Rho, Cdc42, Rab, Arf
-
-
?
additional information
?
-
full-length hemorrhagic toxin TcsH exclusively glucosylates Rho-GTPases. The recombinant transferase domain glucosylates preferably Rho-GTPases but also Ras-GTPases to some extent. Vero cells treated with full length TcsH also show glucosylation of Ras, albeit to a lower extent than of Rho-GTPases
-
-
?
additional information
?
-
full-length hemorrhagic toxin TcsH exclusively glucosylates Rho-GTPases. The recombinant transferase domain glucosylates preferably Rho-GTPases but also Ras-GTPases to some extent. Vero cells treated with full length TcsH also show glucosylation of Ras, albeit to a lower extent than of Rho-GTPases
-
-
?
additional information
?
-
-
full-length hemorrhagic toxin TcsH exclusively glucosylates Rho-GTPases. The recombinant transferase domain glucosylates preferably Rho-GTPases but also Ras-GTPases to some extent. Vero cells treated with full length TcsH also show glucosylation of Ras, albeit to a lower extent than of Rho-GTPases
-
-
?
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UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
UDP-alpha-D-glucose + H-Ras
UDP + D-glucosyl-H-Ras
-
-
-
?
UDP-alpha-D-glucose + human Ras-GTPase
UDP + D-glucosyl-[human Ras-GTPase]
activity of toxin TcsL in human epithelial colorectal adenocarcinoma Caco-2 cells
-
-
?
UDP-alpha-D-glucose + Rac GTPase
UDP + D-glucosyl-[Rac GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Rac1 GTPase
UDP + D-glucosyl-[Rac1 GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Ral GTPase
UDP + D-glucosyl-[Ral GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Rap GTPase
UDP + D-glucosyl-[Rap GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + Ras GTPase
UDP + D-glucosyl-[Ras GTPase]
murine host substrate
-
-
?
UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
-
-
-
?
UDP-alpha-D-glucose + a small GTPase
UDP + D-glucosyl-[a small GTPase]
-
-
-
-
?
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malfunction
the difference in cellular Rac1 and Ras glucosylation when the autoprocessing activity is ablated (mutation C698A) suggests that there is a localization difference for Rac1 and Ras. Mutations in the GTD membrane localization domain inhibit TcsL cytotoxicity
physiological function
glucosylation of Ras by lethal toxin results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway
physiological function
maturation of the host cell endosome causes a conformational change in the pore-forming domain of TcsL, causing it to form a pore in the endosomal membrane. The autoprocessing domain is activated by host inositol hexakisphosphate and cleaves the glucosyltransferase domain (GTD), presumably to permit access to substrates residing at the plasma membrane. The GTD glucosylates small GTPases, predominately Rac, Ras, Ral, and Rap. The glucosylation leads to cytoskeletal rearrangement and rounding of the cells and also causes the induction of apoptosis. Lethal-toxin TcsL shows cytotoxicity in murine pulmonary microvascular endothelial cells (mPMVECs) as model cells, incubation at 33°C or 37°C. Cell viability is determined by CellTiter-Glo luciferase 24 h after intoxication. TcsL autoprocessing and glucosyltransferase activities are important for cytotoxicity, TcsL cytotoxicity is dependent upon GTD localization to the cell membrane. The membrane localization domain (MLD) interaction with the membrane is important for the glucosylation of both Rac1 and Ras
physiological function
quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL), performed by using multiple reaction monitoring (MRM) mass spectrometry
physiological function
full-length hemorrhagic toxin TcsH exclusively glucosylates Rho-GTPases. The recombinant transferase domain glucosylates preferably Rho-GTPases but also Ras-GTPases to some extent. Vero cells treated with full length TcsH also show glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. In addition, enzyme induces a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization
physiological function
isoform TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol. TcsL-cat binds to brain phosphatidylserine-containing membranes and to phosphatidic acid and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. The lipid unsaturation status influences TcsL-cat binding to phospholipids, phosphatidylserines with unsaturated acyl chains and phosphatidic acids with saturated acyl chains being the preferred binding substrates. The phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). Recombinant TcsL-1-93 binds to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively binds to phosphatidylserine and phosphatidic acid
physiological function
the phosphatidylserine binding site is localized on the tip of the N-terminal four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional phosphatidylserine-binding site is required for N-terminal glucosylating domain TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to phosphatidylserine facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution
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C698A
site-directed mutagenesis of the autoprocessing domain, mutant TcsL C698A is able to quickly glucosylate Rac1, similar to wild-type TcsL, but is attenuated in its ability to glucosylate Ras GTPases. The introduction of the autoprocessing mutation does not impact the glucosylation of Rac1 or H-Ras in an in vitro assay
F17N/R18A
site-directed mutagenesis in the GTD membrane localization domain, on the surface of the membrane localization domain (MLD), the mutant shows a defect in membrane association in a liposome binding assay
F17N/R18A/C698A
site-directed mutagenesis in the GTD membrane localization domain, on the surface of the membrane localization domain (MLD), the mutant shows a defect in membrane association in a liposome binding assay. The triple mutant is also inhibited in both Rac1 and Ras glucosylation
I383S/Q385A
mutation allow modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduces the acceptance of UDP-glucose as a donor for glycosylation
F17K
mutation strongly decreases binding to brain phosphatidylserine
K11I
mutation moderately decreases binding to brain phosphatidylserine
K16I
mutation moderately decreases binding to brain phosphatidylserine
Q10A
mutation does not significantly decrease binding to brain phosphatidylserine
Q20A
mutation moderately decreases binding to brain phosphatidylserine
R18P
mutation strongly decreases binding to brain phosphatidylserine
R68A
mutation strongly decreases binding to brain phosphatidylserine
S38A
mutation does not significantly decrease binding to brain phosphatidylserine
V15S
mutation strongly decreases binding to brain phosphatidylserine
Y14A
mutation strongly decreases binding to brain phosphatidylserine
Y78A
mutation does not significantly decrease binding to brain phosphatidylserine
additional information
when glucosyltransferase-deficient TcsL mutant, TcsL DxD, is used to intoxicate cells, the loss of the glucosyltransferase activity renders the mutant unable to glucosylate both Rac1 and Ras
additional information
-
when glucosyltransferase-deficient TcsL mutant, TcsL DxD, is used to intoxicate cells, the loss of the glucosyltransferase activity renders the mutant unable to glucosylate both Rac1 and Ras
additional information
the Dxd motif, i.e. residues D286-D288, is required at least for activation of the UDP-glucose hydrolysis activity by Mn2+
additional information
-
the Dxd motif, i.e. residues D286-D288, is required at least for activation of the UDP-glucose hydrolysis activity by Mn2+
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Just, I.; Selzer, J.; Hofmann, F.; Green, G. A.; Aktories, K.
Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation
J. Biol. Chem.
271
10149-10153
1996
Paeniclostridium sordellii (Q46342), Paeniclostridium sordellii 6018 (Q46342)
brenda
Jank, T.; Reinert, D.; Giesemann, T.; Schulz, G.; Aktories, K.
Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis
J. Biol. Chem.
280
37833-37838
2005
Clostridioides difficile (P18177), Clostridioides difficile, Clostridioides difficile VPI 10463 (P18177), Clostridium novyi (Q46149), Clostridium novyi, Clostridium novyi 19402 (Q46149), Paeniclostridium sordellii (Q46342), Paeniclostridium sordellii, Paeniclostridium sordellii 6018 (Q46342)
brenda
Ziegler, M.O.; Jank, T.; Aktories, K.; Schulz, G.E.
Conformational changes and reaction of clostridial glycosylating toxins
J. Mol. Biol.
377
1346-1356
2007
Clostridium novyi (Q46149), Paeniclostridium sordellii (Q46342)
brenda
Genth, H.; Pauillac, S.; Schelle, I.; Bouvet, P.; Bouchier, C.; Varela-Chavez, C.; Just, I.; Popoff, M.R.
Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins
Cell. Microbiol.
16
1706-1721
2014
Paeniclostridium sordellii (M9ZTT7), Paeniclostridium sordellii (V5T923), Paeniclostridium sordellii, Paeniclostridium sordellii vpi9048 (M9ZTT7), Paeniclostridium sordellii vpi9048 (V5T923)
brenda
Varela Chavez, C.; Hoos, S.; Haustant, G.M.; Chenal, A.; England, P.; Blondel, A.; Pauillac, S.; Lacy, D.B.; Popoff, M.R.
The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid
Cell. Microbiol.
17
1477-1493
2015
Paeniclostridium sordellii (V5T923), Paeniclostridium sordellii
brenda
Varela Chavez, C.; Haustant, G.M.; Baron, B.; England, P.; Chenal, A.; Pauillac, S.; Blondel, A.; Popoff, M.R.
The tip of the four N-terminal alpha-helices of Clostridium sordellii lethal toxin contains the interaction site with membrane phosphatidylserine facilitating small GTPases glucosylation
Toxins
8
90
2016
Paeniclostridium sordellii (V5T923), Paeniclostridium sordellii
brenda
Thiele, T.L.; Stuber, T.P.; Hauer, P.J.
Detection of Clostridium sordellii strains expressing hemorrhagic toxin (TcsH) and implications for diagnostics and regulation of veterinary vaccines
Vaccine
31
5082-5087
2013
Paeniclostridium sordellii (M9ZTT7), Paeniclostridium sordellii (V5T923), Paeniclostridium sordellii, Paeniclostridium sordellii VPI 9048 (M9ZTT7), Paeniclostridium sordellii VPI 9048 (V5T923)
brenda
Genth, H.; Schelle, I.; Just, I.
Metal ion activation of Clostridium sordellii lethal toxin and Clostridium difficile toxin B
Toxins
8
109
2016
Clostridioides difficile (P18177), Clostridioides difficile, Paeniclostridium sordellii (V5T923), Paeniclostridium sordellii
brenda
Genth, H.; Junemann, J.; Laemmerhirt, C.M.; Luecke, A.C.; Schelle, I.; Just, I.; Gerhard, R.; Pich, A.
Difference in mono-O-glucosylation of Ras subtype GTPases between toxin A and toxin B from Clostridioides difficile strain 10463 and lethal toxin from Clostridium sordellii strain 6018
Front. Microbiol.
9
3078
2018
Clostridioides difficile (P16154), Clostridioides difficile (P18177), Clostridioides difficile, Clostridioides difficile 10463 (P16154), Clostridioides difficile 10463 (P18177), Paeniclostridium sordellii (Q46342), Paeniclostridium sordellii, Paeniclostridium sordellii 6018 (Q46342)
brenda
Craven, R.; Lacy, D.
Clostridium sordellii lethal-toxin autoprocessing and membrane localization activities drive GTPase glucosylation profiles in endothelial cells
mSphere
1
e00012-15
2016
Paeniclostridium sordellii (Q46342), Paeniclostridium sordellii, Paeniclostridium sordellii JGS6382 (Q46342)
brenda